In the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion, because an ectopic neomycin phosphortransferase positive selection cassette (NEO) may cause a position effect. During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-Time PCR using both SYBR Green and specific minor groove binder (MGB) probes to evaluate the copies of NEO using the comparative delta-delta Ct method versus the Rpp30 reference gene

Brusco, Alfredo

Brussino, Alessandro

Mancini, Cecilia

Messana, Erika

Turco, Emilia

English

PubMed

Cecilia Mancini

Erika Messana

Emilia Turco

Alessandro Brussino

Alfredo Brusco

Springer - Publisher Connector

SHORT REPORT Open Access
Gene-targeted embryonic stem cells: real-time
PCR assay for estimation of the number of
neomycin selection cassettes
Cecilia Mancini1, Erika Messana1, Emilia Turco1,3, Alessandro Brussino1 and Alfredo Brusco1,2*
Abstract
In the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion,
because an ectopic neomycin phosphortransferase positive selection cassette (NEO) may cause a position effect.
During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-Time
PCR using both SYBR Green and specific minor groove binder (MGB) probes to evaluate the copies of NEO using
the comparative delta-delta Ct method versus the Rpp30 reference gene.
We compared the results from Southern blot, routinely used to quantify NEO copies, with the two Real-Time PCR
assays. Twenty-two clones containing the single NEO copy showed values of 0.98 ± 0.24 (mean ± 2 S.D.), and were
clearly distinguishable from clones with two or more NEO copies.
This method was found to be useful, easy, sensitive and fast and could substitute for the widely used, but
laborious Southern blot method.
Two methods are available for the introduction and
modification of mouse genomic DNA sequences: (i)
microinjection of one or more transgenes into the pro-
nucleus of a fertilized mouse oocyte, which usually leads
to random incorporation into the genome as head-to-
tail concatamers of 1-1000 units, or (ii) the use of con-
structs that undergo a site-specific recombination in
embryonic stem cells (ES) in order to disrupt the func-
tion of a target gene (knockout) or to mutate a gene
(knockin). Modified ES cells are then injected into the
blastocyst [1]. In the latter case, the production of
knockout or knockin ES cells is obtained through gene
targeting by homologous recombination. In this work,
ES cells were transfected by electroporation with a con-
struct containing a specific genomic sequence harbour-
ing the required mutation, along with the neomycin
phophortransferase positive selection cassette (NEO) for
selection of positive recombinants, flanked by two
homology sequences ("arms”) driving the recombination
[2,3]. Homologous recombination occurs in a small
number of transfected cells, resulting in the introduction
of the mutation present in the targeting construct into
the gene of interest. However, despite the presence of
the two “arms”, there may be a variable number of ran-
dom integrations that may cause a position effect [4-6].
To identify the mutant ES cell clones to be microin-
jected, two Southern blots are usually performed: one to
detect ES clones in which homologous recombination
has occurred, and the other to verify the number of
NEO cassettes. Usually between two and three hundred
clones are analysed: useful clones are routinely just 1 -
2% of the total. This low percentage is mainly due to
the event of the vector being inserted in ectopic sites.
One member of our group is responsible for a facility
within the Molecular Biotechnology Center in Torino,
aimed at the preparation of transgenic mice using
recombinant ES cells. In routine work, it became neces-
sary to have a rapid test to exclude the presence of addi-
tional copies of the NEO cassette in ES clones in which
homologous recombination was successfully obtained.
Here we describe a screening method using a rapid
semi-quantitative real-time PCR, which was validated on
ES clones with different NEO copies (0, 1, 2, > 2 copies),
previously assessed by Southern blot.
From one of the projects involving the preparation of
recombinant mice, we selected 45 genomic DNA
* Correspondence: alfredo.brusco@unito.it
1Department of Genetics, Biology and Biochemistry, University of Torino,
Torino, Italy
Full list of author information is available at the end of the article
Mancini et al. Biological Procedures Online 2011, 13:10
http://www.biologicalproceduresonline.com/content/13/1/10 Biological Procedures
Online
© 2011 Mancini et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
extracted from ES clones that then underwent Southern
blot screening. DNA extraction was performed using
standard phenol-chloroform method [7]. Southern blot
was performed using standard conditions for gel run,
transfer and hybridisation. A NEO probe of 773 bp was
used to evaluate the number of transgenic plasmid
insertions.
As an alternative or complementary method to assess
the NEO cassette copy number, we set up an assay based
on quantitative real-time PCR (qPCR). The assay was
performed with two protocols on an ABI 7500 Fast
instrument (Applied Biosystems, Foster City, CA, USA);
data were analyzed using the 7500 software. The two
approaches were: (i) an MGB-based assay (MGB-assay),
and (ii) a SYBR Green-based assay (SYBR-assay) (Applied
Biosystems). Using the Primer Express software (Applied
Biosystems) we designed specific PCR primers to amplify
62 bp of highly conserved sequences between two
different NEO cassette-containing plasmids PL451 and
PL452 (http://web.ncifcrf.gov/research/brb/recombineer-
ingInformation.aspx). The mouse Rpp30 gene (63 bp,
RefSeq NM_019428.3) was used as a reference gene. This
gene is orthologous to the human RNaseP, widely used as
a copy number reference in qPCR assay. Real-time
PCR conditions and primer sequences are reported in
Figure 1.
In the MGB-assay we combined the two NEO and
Rpp30 assays in a duplex PCR including two internal
Taqman-MGB probes (5’-FAM labelled for NEO and 5’-
VIC labelled for Rpp30) (Figure 1). Each sample was
consistently run in triplicate with a blank well to check
for contaminations.
In the SYBR-assay the two reactions were run in sepa-
rate wells, using a Fast SYBR green mix (Applied Biosys-
tems). The efficiency of each assay was verified with a
standard curve starting from 100 ng of mouse DNA, using
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Figure 1 Real-time PCR protocols for MGB- (left) and SYBR-assay (right). Protocols and run conditions that were used for MGB- and SYBR-
assays are described in the upper part. Below are the oligo sequences and oligo concentrations used to prepare the NEO and Rpp30 assays. For
the MGB-assay we prepared two (20×) mixes, one (*) for the target gene (NEO) and one (**) for the reference gene (Rpp30). For both assays, the
final concentration in each reaction was 900 nM for oligos, 250 nM for probes (MGB-assay only). The MGB assay was prepared using TaqMan 2×
Master Mix (Applied Biosystems): a standard run protocol of two hours was used. The SYBR-assay was performed with Fast SYBR Green 2×
Master Mix (Applied Biosystems); the fast protocol reported took only 40 min.
Mancini et al. Biological Procedures Online 2011, 13:10
http://www.biologicalproceduresonline.com/content/13/1/10
Page 2 of 4
four serial dilutions from 1:1 to 1:8. The MGB-assay had
80% efficiency, whereas SYBR-assay gave 93% efficiency.
Gene copy-number was calculated using the comparative
delta-delta Ct method [8]. In each experiment, we normal-
ized the ΔCt of the sample to the mean ΔCt of three ES
clones with a single NEO copy verified using Southern
blot; these values (named nNEO) were expected to be ~
“1” in the case of a single copy of NEO, ~ “2” in the case of
two copies of NEO, and “n” for “n” copies of NEO.
Using Southern blot, twenty-two of the 45 ES clones
showed one copy of NEO, and 23 clones had multiple
NEO copies. We analyzed all samples using both of the
two real-time PCR assays (Figure 2). The 22 clones con-
taining a single NEO copy showed a nNEO value of 0.98
± 0.24 (mean ± 2 S.D.) (range 0.81-1.20) for the MGB-
assay and 1.01 ± 0.24 (range 0.80-1.24) for the SYBR-
assay (Figure 2A). As expected for a single copy of
NEO, the value of nNEO was between 0.8 and 1.2. In a
Figure 2 Quantitative real-time PCR validation and results for NEO copy number determination, using MGB-assay (in red) and SYBR-
assay (in green). (A) Twenty-two ES clones (abscissa, numbers from 1 to 22) showed a nNEO value between 0.8 and 1.2 (ordinate). One
genomic DNA from a homozygous knockout mouse was used as control (CTRL 2NEO, nNEO = 2.2). Error bars indicate triplicates delta-delta Ct
standard deviations. (B) Real-time PCR results: 45 ES clones are plotted on the graph, for each assay using a log2 scale. The dotted line indicates
a nNEO threshold of 1.2, the upper cutoff for considering the presence of a single NEO copy. Samples tested with either of the two assays had
similar nNEO values, as shown by the similar population plots.
Mancini et al. Biological Procedures Online 2011, 13:10
http://www.biologicalproceduresonline.com/content/13/1/10
Page 3 of 4
control DNA with two NEO copies, the value of nNEO
was 2.2 for the MGB-assay and 2.4 for the SYBR-assay
(Figure 2A).
The remaining 23 ES clones (≥ 2 NEO copies) showed
a variable number of nNEO from ~2 to > 60 (Figure
2B). Comparing results of the two qPCR assays for the
same sample, we saw that the values of nNEO had a
minimal variability. Although the nNEO number prob-
ably reflects the amount of cassettes, calculating their
exact number was beyond our scope. Curiously, we
found high copy-number insertions that reached up to
60; this could be explained by the presence of concata-
mers of the vector, rather than multiple insertions in
the mouse genome.
Southern blot analysis has long been the reference
method for the detection of NEO copy number in ES
clones. However, this method requires large amounts of
DNA samples, as well as being laborious, time-consum-
ing, and sometimes difficult to interpret. Our experience
in transgenic mouse model preparation has led to the
conclusion that having an alternative method is highly
favourable. We propose a semi-quantitative real-time
PCR method, that only requires small amounts of DNA,
and much less time to be performed (the Fast, SYBR-
assay format takes under one hour). This method
appears to be sensitive enough even for identification of
single NEO insertions. Of the two assays tested, the
MGB-assay is the least convenient, as it requires two
specific and expensive fluorescently-labelled probes, and
does not show any practical advantage over the SYBR-
assay.
Approaches based on real-time PCR to validate ES
positive clones have been previously described, using
absolute quantification of NEO, or a relative quantifica-
tion to detect deletion of the target gene [9,10]. Our
method, based on a relative PCR quantification, can be
easily reproduced in other laboratories using different
technical platforms, and does not need the preparation
of standards.
One possible drawback may be the genomic DNA
quality/degradation that needs to be checked in case of
non-reproducible data.
In conclusion, our real-time PCR assay to quantify
NEO copy number is a valid alternative tool to Southern
blot for the rapid screening of large numbers of ES cell
clones during the production of knockout or knockin
mouse models.
Acknowledgements
This work was supported by Telethon research grant GGP07110 to AB.
Author details
1Department of Genetics, Biology and Biochemistry, University of Torino,
Torino, Italy. 2S.C.D.U. Medical Genetics, A.O.U. San Giovanni Battista, Torino,
Italy. 3Molecular Biotechnology Center, Torino, Italy.
Authors’ contributions
CM and EM carried out laboratory experiments, analyzed data and drafted
the manuscript; ET analyzed data and revised the manuscript; AB and AB
designed the experiments, analyzed data, drafted and revised the
manuscript.
All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 29 July 2011 Accepted: 28 October 2011
Published: 28 October 2011
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doi:10.1186/1480-9222-13-10
Cite this article as: Mancini et al.: Gene-targeted embryonic stem cells:
real-time PCR assay for estimation of the number of neomycin selection
cassettes. Biological Procedures Online 2011 13:10.
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Gene-targeted embryonic stem cells: real-time PCR assay for estimation of the number of neomycin selection cassettes

Abstract In the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion, because an ectopic neomycin phosphortransferase positive selection cassette (NEO) may cause a position effect. During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-Time PCR using both SYBR Green and specific minor groove binder (MGB) probes to evaluate the copies of NEO using the comparative delta-delta Ct method versus the Rpp30 reference gene. We compared the results from Southern blot, routinely used to quantify NEO copies, with the two Real-Time PCR assays. Twenty-two clones containing the single NEO copy showed values of 0.98 ± 0.24 (mean ± 2 S.D.), and were clearly distinguishable from clones with two or more NEO copies. This method was found to be useful, easy, sensitive and fast and could substitute for the widely used, but laborious Southern blot method.</p

Mancini Cecilia

Messana Erika

Turco Emilia

Brussino Alessandro

Brusco Alfredo

Directory of Open Access Journals

Biological Procedures Online

Institutional Research Information System University of Turin

SHORT REPORT Open AccessGene-targeted embryonic stem cells: real-timePCR assay for estimation of the number ofneomycin selection cassettesCecilia Mancini1, Erika Messana1, Emilia Turco1,3, Alessandro Brussino1 and Alfredo Brusco1,2*AbstractIn the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion,because an ectopic neomycin phosphortransferase positive selection cassette (NEO) may cause a position effect.During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-TimePCR using both SYBR Green and specific minor groove binder (MGB) probes to evaluate the copies of NEO usingthe comparative delta-delta Ct method versus the Rpp30 reference gene.We compared the results from Southern blot, routinely used to quantify NEO copies, with the two Real-Time PCRassays. Twenty-two clones containing the single NEO copy showed values of 0.98 ± 0.24 (mean ± 2 S.D.), and wereclearly distinguishable from clones with two or more NEO copies.This method was found to be useful, easy, sensitive and fast and could substitute for the widely used, butlaborious Southern blot method.Two methods are available for the introduction andmodification of mouse genomic DNA sequences: (i)microinjection of one or more transgenes into the pro-nucleus of a fertilized mouse oocyte, which usually leadsto random incorporation into the genome as head-to-tail concatamers of 1-1000 units, or (ii) the use of con-structs that undergo a site-specific recombination inembryonic stem cells (ES) in order to disrupt the func-tion of a target gene (knockout) or to mutate a gene(knockin). Modified ES cells are then injected into theblastocyst [1]. In the latter case, the production ofknockout or knockin ES cells is obtained through genetargeting by homologous recombination. In this work,ES cells were transfected by electroporation with a con-struct containing a specific genomic sequence harbour-ing the required mutation, along with the neomycinphophortransferase positive selection cassette (NEO) forselection of positive recombinants, flanked by twohomology sequences ("arms”) driving the recombination[2,3]. Homologous recombination occurs in a smallnumber of transfected cells, resulting in the introductionof the mutation present in the targeting construct intothe gene of interest. However, despite the presence ofthe two “arms”, there may be a variable number of ran-dom integrations that may cause a position effect [4-6].To identify the mutant ES cell clones to be microin-jected, two Southern blots are usually performed: one todetect ES clones in which homologous recombinationhas occurred, and the other to verify the number ofNEO cassettes. Usually between two and three hundredclones are analysed: useful clones are routinely just 1 -2% of the total. This low percentage is mainly due tothe event of the vector being inserted in ectopic sites.One member of our group is responsible for a facilitywithin the Molecular Biotechnology Center in Torino,aimed at the preparation of transgenic mice usingrecombinant ES cells. In routine work, it became neces-sary to have a rapid test to exclude the presence of addi-tional copies of the NEO cassette in ES clones in whichhomologous recombination was successfully obtained.Here we describe a screening method using a rapidsemi-quantitative real-time PCR, which was validated onES clones with different NEO copies (0, 1, 2, > 2 copies),previously assessed by Southern blot.From one of the projects involving the preparation ofrecombinant mice, we selected 45 genomic DNA* Correspondence: alfredo.brusco@unito.it1Department of Genetics, Biology and Biochemistry, University of Torino,Torino, ItalyFull list of author information is available at the end of the articleMancini et al. Biological Procedures Online 2011, 13:10http://www.biologicalproceduresonline.com/content/13/1/10 Biological ProceduresOnline© 2011 Mancini et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.extracted from ES clones that then underwent Southernblot screening. DNA extraction was performed usingstandard phenol-chloroform method [7]. Southern blotwas performed using standard conditions for gel run,transfer and hybridisation. A NEO probe of 773 bp wasused to evaluate the number of transgenic plasmidinsertions.As an alternative or complementary method to assessthe NEO cassette copy number, we set up an assay basedon quantitative real-time PCR (qPCR). The assay wasperformed with two protocols on an ABI 7500 Fastinstrument (Applied Biosystems, Foster City, CA, USA);data were analyzed using the 7500 software. The twoapproaches were: (i) an MGB-based assay (MGB-assay),and (ii) a SYBR Green-based assay (SYBR-assay) (AppliedBiosystems). Using the Primer Express software (AppliedBiosystems) we designed specific PCR primers to amplify62 bp of highly conserved sequences between twodifferent NEO cassette-containing plasmids PL451 andPL452 (http://web.ncifcrf.gov/research/brb/recombineer-ingInformation.aspx). The mouse Rpp30 gene (63 bp,RefSeq NM_019428.3) was used as a reference gene. Thisgene is orthologous to the human RNaseP, widely used asa copy number reference in qPCR assay. Real-timePCR conditions and primer sequences are reported inFigure 1.In the MGB-assay we combined the two NEO andRpp30 assays in a duplex PCR including two internalTaqman-MGB probes (5’-FAM labelled for NEO and 5’-VIC labelled for Rpp30) (Figure 1). Each sample wasconsistently run in triplicate with a blank well to checkfor contaminations.In the SYBR-assay the two reactions were run in sepa-rate wells, using a Fast SYBR green mix (Applied Biosys-tems). The efficiency of each assay was verified with astandard curve starting from 100 ng of mouse DNA, using???????????????????????? ?? ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ?????????????????????????????????????????????????????????????????????????????????????????????? ?????????? ?????? ??????????????????? ?????????????????????? ????????????????????????? ???????????????? ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????Figure 1 Real-time PCR protocols for MGB- (left) and SYBR-assay (right). Protocols and run conditions that were used for MGB- and SYBR-assays are described in the upper part. Below are the oligo sequences and oligo concentrations used to prepare the NEO and Rpp30 assays. Forthe MGB-assay we prepared two (20×) mixes, one (*) for the target gene (NEO) and one (**) for the reference gene (Rpp30). For both assays, thefinal concentration in each reaction was 900 nM for oligos, 250 nM for probes (MGB-assay only). The MGB assay was prepared using TaqMan 2×Master Mix (Applied Biosystems): a standard run protocol of two hours was used. The SYBR-assay was performed with Fast SYBR Green 2×Master Mix (Applied Biosystems); the fast protocol reported took only 40 min.Mancini et al. Biological Procedures Online 2011, 13:10http://www.biologicalproceduresonline.com/content/13/1/10Page 2 of 4four serial dilutions from 1:1 to 1:8. The MGB-assay had80% efficiency, whereas SYBR-assay gave 93% efficiency.Gene copy-number was calculated using the comparativedelta-delta Ct method [8]. In each experiment, we normal-ized the ΔCt of the sample to the mean ΔCt of three ESclones with a single NEO copy verified using Southernblot; these values (named nNEO) were expected to be ~“1” in the case of a single copy of NEO, ~ “2” in the case oftwo copies of NEO, and “n” for “n” copies of NEO.Using Southern blot, twenty-two of the 45 ES clonesshowed one copy of NEO, and 23 clones had multipleNEO copies. We analyzed all samples using both of thetwo real-time PCR assays (Figure 2). The 22 clones con-taining a single NEO copy showed a nNEO value of 0.98± 0.24 (mean ± 2 S.D.) (range 0.81-1.20) for the MGB-assay and 1.01 ± 0.24 (range 0.80-1.24) for the SYBR-assay (Figure 2A). As expected for a single copy ofNEO, the value of nNEO was between 0.8 and 1.2. In aFigure 2 Quantitative real-time PCR validation and results for NEO copy number determination, using MGB-assay (in red) and SYBR-assay (in green). (A) Twenty-two ES clones (abscissa, numbers from 1 to 22) showed a nNEO value between 0.8 and 1.2 (ordinate). Onegenomic DNA from a homozygous knockout mouse was used as control (CTRL 2NEO, nNEO = 2.2). Error bars indicate triplicates delta-delta Ctstandard deviations. (B) Real-time PCR results: 45 ES clones are plotted on the graph, for each assay using a log2 scale. The dotted line indicatesa nNEO threshold of 1.2, the upper cutoff for considering the presence of a single NEO copy. Samples tested with either of the two assays hadsimilar nNEO values, as shown by the similar population plots.Mancini et al. Biological Procedures Online 2011, 13:10http://www.biologicalproceduresonline.com/content/13/1/10Page 3 of 4control DNA with two NEO copies, the value of nNEOwas 2.2 for the MGB-assay and 2.4 for the SYBR-assay(Figure 2A).The remaining 23 ES clones (≥ 2 NEO copies) showeda variable number of nNEO from ~2 to > 60 (Figure2B). Comparing results of the two qPCR assays for thesame sample, we saw that the values of nNEO had aminimal variability. Although the nNEO number prob-ably reflects the amount of cassettes, calculating theirexact number was beyond our scope. Curiously, wefound high copy-number insertions that reached up to60; this could be explained by the presence of concata-mers of the vector, rather than multiple insertions inthe mouse genome.Southern blot analysis has long been the referencemethod for the detection of NEO copy number in ESclones. However, this method requires large amounts ofDNA samples, as well as being laborious, time-consum-ing, and sometimes difficult to interpret. Our experiencein transgenic mouse model preparation has led to theconclusion that having an alternative method is highlyfavourable. We propose a semi-quantitative real-timePCR method, that only requires small amounts of DNA,and much less time to be performed (the Fast, SYBR-assay format takes under one hour). This methodappears to be sensitive enough even for identification ofsingle NEO insertions. Of the two assays tested, theMGB-assay is the least convenient, as it requires twospecific and expensive fluorescently-labelled probes, anddoes not show any practical advantage over the SYBR-assay.Approaches based on real-time PCR to validate ESpositive clones have been previously described, usingabsolute quantification of NEO, or a relative quantifica-tion to detect deletion of the target gene [9,10]. Ourmethod, based on a relative PCR quantification, can beeasily reproduced in other laboratories using differenttechnical platforms, and does not need the preparationof standards.One possible drawback may be the genomic DNAquality/degradation that needs to be checked in case ofnon-reproducible data.In conclusion, our real-time PCR assay to quantifyNEO copy number is a valid alternative tool to Southernblot for the rapid screening of large numbers of ES cellclones during the production of knockout or knockinmouse models.AcknowledgementsThis work was supported by Telethon research grant GGP07110 to AB.Author details1Department of Genetics, Biology and Biochemistry, University of Torino,Torino, Italy. 2S.C.D.U. Medical Genetics, A.O.U. San Giovanni Battista, Torino,Italy. 3Molecular Biotechnology Center, Torino, Italy.Authors’ contributionsCM and EM carried out laboratory experiments, analyzed data and draftedthe manuscript; ET analyzed data and revised the manuscript; AB and ABdesigned the experiments, analyzed data, drafted and revised themanuscript.All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 29 July 2011 Accepted: 28 October 2011Published: 28 October 2011References1. Thomas KR, Deng C, Capecchi MR: High-fidelity gene targeting inembryonic stem cells by using sequence replacement vectors. Mol CellBiol 1992, 12:2919-2923.2. Meier ID, Bernreuther C, Tilling T, Neidhardt J, Wong YW, Schulze C,Streichert T, Schachner M: Short DNA sequences inserted for genetargeting can accidentally interfere with off-target gene expression.FASEB J 2010, 24:1714-1724.3. Joyner A: Gene targeting a practical approach New York: Oxford UniversityPress; 2000.4. Olson EN, Arnold HH, Rigby PW, Wold BJ: Know your neighbors: threephenotypes in null mutants of the myogenic bHLH gene MRF4. Cell1996, 85:1-4.5. Rijli FM, Dolle P, Fraulob V, LeMeur M, Chambon P: Insertion of a targetingconstruct in a Hoxd-10 allele can influence the control of Hoxd-9expression. Dev Dyn 1994, 201:366-377.6. Pham CT, MacIvor DM, Hug BA, Heusel JW, Ley TJ: Long-range disruptionof gene expression by a selectable marker cassette. Proc Natl Acad SciUSA 1996, 93:13090-13095.7. Sambrook J, Russell D: Molecular Cloning: A Laboratory Manual Cold SpringHarbor Laboratory Press; 2001.8. Livak KJ, Schmittgen TD: Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods2001, 25:402-408.9. Valenzuela DM, Murphy AJ, Frendewey D, Gale NW, Economides AN,Auerbach W, Poueymirou WT, Adams NC, Rojas J, Yasenchak J, et al: High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 2003, 21:652-659.10. Coumoul X, Shukla V, Li C, Wang RH, Deng CX: Conditional knockdown ofFgfr2 in mice using Cre-LoxP induced RNA interference. Nucleic Acids Res2005, 33:e102.doi:10.1186/1480-9222-13-10Cite this article as: Mancini et al.: Gene-targeted embryonic stem cells:real-time PCR assay for estimation of the number of neomycin selectioncassettes. Biological Procedures Online 2011 13:10.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitMancini et al. Biological Procedures Online 2011, 13:10http://www.biologicalproceduresonline.com/content/13/1/10Page 4 of 4

Gene-targeted embryonic stem cells: real-time PCR assay for estimation of the number of neomycin selection cassettes.

al: Highthroughput engineering of the mouse genome coupled with highresolution expression analysis. Nat Biotechnol

Capecchi MR: High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors. Mol Cell Biol

Chambon P: Insertion of a targeting construct in a Hoxd-10 allele can influence the control of Hoxd-9 expression. Dev Dyn

Gene targeting a practical approach

Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell

Molecular Cloning:

Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods

Short DNA sequences inserted for gene targeting can accidentally interfere with off-target gene expression.

TJ: Long-range disruption of gene expression by a selectable marker cassette.

https://iris.unito.it/bitstream/2318/96327/1/70.Gene%20targeted%20embryonic%20stem%20cells%20real-time%20PCR%20to%20estimate%20NEO%20cassette_2011.pdf

Gene-targeted embryonic stem cells: real-time PCR assay for estimation of the number of neomycin selection cassettes

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